This invention relates to apparatus for processing samples. More particularly, the present invention relates to automated apparatus and methods for washing supports carrying biological material such as polynucleotides. The invention has particular application to the area of analyzing the results of hybridization reactions involving nucleic acids.
Determining the nucleotide sequences and expression levels of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship, for example, to disease discovery and disease management. Analysis of genetic information plays a crucial role in biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. Such a determination permits the early detection of infectious organisms such as bacteria, viruses, etc.; genetic diseases such as sickle cell anemia; and various cancers. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.
Unique or polymorphic nucleotide sequences in a polynucleotide can be detected by hybridization with an oligonucleotide probe. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. An oligonucleotide probe employed in the detection is selected with a nucleotide sequence complementary, usually exactly complementary, to the nucleotide sequence in the target nucleic acid. Following hybridization of the probe with the target nucleic acid, any oligonucleotide probe/nucleic acid hybrids that have formed are typically separated from unhybridized probe. The amount of oligonucleotide probe in either of the two separated media is then tested to provide a qualitative or quantitative measurement of the amount of target nucleic acid originally present. In surface-bound DNA arrays, this separation is typically accomplished by washing the unbound and non-specifically bound material away from the array surface. The resulting wash protocol is normally optimized to the specific requirements of the assay, the probe type, the surface selected and other considerations. The surface is then scanned for the presence of the target.
Direct detection of labeled target nucleic acid hybridized to surface-bound polynucleotide probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations. The arrays may be microarrays created by in-situ synthesis or oligonucleotide deposition. Microarrays created by cDNA deposition are used to analyze gene expression patterns and perform genome scanning. Protein arrays are very useful for determining the presence and quantity of specific proteins in a cell or tissue. These arrays may have either proteins or aptamers bound to the surface. Due to the large number of genes in the human genome and other mammals and plants and the large number of proteins created, it is desirable to automate this hybridization process on microarrays.
In one approach, cell matter is lysed, to release its DNA, mRNA or protein which are then separated out by electrophoresis or other means and amplified, if necessary and then tagged with a fluorescent or other label. The resulting mix is exposed to an array of oligonucleotide, cDNA, aptamer or protein probes, whereupon selective binding to matching probe sites takes place. The array is then washed and interrogated to determine the extent of hybridization reactions. In one approach the array is imaged so as to reveal for analysis and interpretation the sites where binding has occurred.
Biological assays involving fluorescently labeled molecules or scattering structures to detect, quantify or identify target chemical species bound to surfaces often use optical detection and imaging systems. Arrays of different chemical probe species provide methods of highly parallel detection, and hence improved speed and efficiency, in assays. These arrays are, for example, DNA arrays and protein matrix arrays, which need to be scanned to measure the number densities of labeled molecules and hence the concentration of target or probe molecules in solution. This sensing process often is accomplished by means of a fluorescence imaging system. Chemiluminescence and radioisotopes are alternative methods commonly employed.
As mentioned above, usually a mosaic of different probes are individually localized to discrete, known areas of a surface of a support. The support may be utilized and analyzed directly or the support may be part of a package, which houses the support. For example, hybridization arrays may be part of a self-contained package. After a hybridization process occurs on the surface of the support, the surface must be washed to remove the unbound and non-specifically bound sample. In a self-contained microarray package, for example, the test sample in the package needs to be washed in order for the features of the microarray to stand out during the detection process such as a scanning process. During the washing process, it is important for the washing protocol be followed precisely in order to remove any excess sample from the actual surface without damaging or destroying the microarray on the surface of the support or causing the hybridized material to melt off the array, thus losing much or all of the signal obtained during the hybridization process.